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  本文選題：間充質(zhì)干細(xì)胞 + CD11b~+Gr1~+�。� 參考：《北京協(xié)和醫(yī)學(xué)院》2014年碩士論文

【摘要】：背景：間充質(zhì)干細(xì)胞(mesenchymal stem cells,MSCs)是中胚層來源的具有高度自我更新能力和多向分化能力的多能干細(xì)胞,廣泛存在于全身多種組織中,可在體外培養(yǎng)擴增,并能在特定條件下分化為神經(jīng)細(xì)胞、成骨細(xì)胞、軟骨細(xì)胞、心肌細(xì)胞、脂肪細(xì)胞等,是細(xì)胞替代治療和組織工程首選的種子細(xì)胞,具有廣闊的臨床應(yīng)用前景。此外,間充質(zhì)干細(xì)胞的染色體核型和端粒酶活性均穩(wěn)定,不表達(dá)組織相容性復(fù)合物(MHC)等刺激分子,因此不易被宿主T細(xì)胞識別,可逃脫宿主免疫系統(tǒng)排斥,具有低免疫性。MSCs還可通過旁分泌作用抑制B淋巴細(xì)胞和T淋巴細(xì)胞的增殖,誘導(dǎo)T淋巴細(xì)胞的凋亡而起到免疫抑制作用。微環(huán)境中的多種因素兒可影響MSCs的免疫調(diào)節(jié)能力,TNF-a可誘導(dǎo)MSCs高表達(dá)iNOS,通過NO依賴的方式直接抑制免疫應(yīng)答；也可促進(jìn)COX-2和PGE2表達(dá)上調(diào),通過PGE2依賴的方式直接抑制免疫應(yīng)答。另外,MSCs可通過分泌大量的細(xì)胞因子吸引更多的單核細(xì)胞、巨噬細(xì)胞以及中性粒細(xì)胞,從而影響腫瘤的生長。但MSCs對骨髓中CD11b+Grl+細(xì)胞有何影響,這種影響又是如何作用于腫瘤過程的,還有待進(jìn)一步探究。 目的：探討間充質(zhì)干細(xì)胞(MSCs)誘導(dǎo)骨髓來源的CD11b+Grl+細(xì)胞免疫功能對腫瘤的作用。 方法：(1)將小鼠骨髓中分選的CD11b+Gr1+細(xì)胞與MSCs共培養(yǎng)72h后,計數(shù)。應(yīng)用流式細(xì)胞技術(shù)測定活細(xì)胞百分比與細(xì)胞免疫表型。(2)通過T細(xì)胞抑制性實驗檢測與MSCs共培養(yǎng)后CD11b+Grl+細(xì)胞：將與TNF-α刺激過的MSCs或MSCs共培養(yǎng)后CD11b+Grl+細(xì)胞加入抗-CD3抗體刺激的T淋巴細(xì)胞體系中,共培養(yǎng)72h。流式細(xì)胞檢測T淋巴細(xì)胞的增殖和活化。(3)建立4T1腫瘤模型,體內(nèi)檢測與TNF-α刺激過的MSCs或MSCs共培養(yǎng)后CD11b+Grl+細(xì)胞對腫瘤的影響作用。 結(jié)果：MSC能夠促進(jìn)骨髓中CD11b+Grl+細(xì)胞的存活和生成,同時可誘導(dǎo)正常小鼠骨髓中CD11b+Grl+細(xì)胞對T細(xì)胞的抑制作用。動物實驗結(jié)果顯示,MSCs處理后的骨髓CD11b+Grl+細(xì)胞可以促進(jìn)腫瘤的生長,加速小鼠的死亡 結(jié)論：MSCs可以通過促進(jìn)骨髓來源的CD11b+Grl+細(xì)胞的抑制作用加速腫瘤的生長進(jìn)程。 背景：中性粒細(xì)胞大量存在于腫瘤微環(huán)境中,但是它對腫瘤進(jìn)程的影響還未知。中性粒細(xì)胞特定因素的作用下,會發(fā)揮一定的促炎功能。此外,中性粒細(xì)胞可以從不具備免疫抑制功能的表型轉(zhuǎn)換到具有免疫抑制功能的表型：有研究發(fā)現(xiàn)粒細(xì)胞-巨噬細(xì)胞集落刺激因子(GM-CSF)和TGF-β都可以將小鼠骨髓來源的中性粒細(xì)胞轉(zhuǎn)換為具有免疫抑制功能的表型。但是,在癌癥發(fā)展的過程中,中性粒細(xì)胞是如何被轉(zhuǎn)換為免疫抑制表型,從而促進(jìn)腫瘤發(fā)展的,我們還知之甚少。 間充質(zhì)干細(xì)胞(Mesenchymal stem cell, MSCs)是骨髓中一類具有再生能力的基質(zhì)細(xì)胞,并作為骨髓造血干細(xì)胞的支持細(xì)胞支持造血,同時MSCs也具有免疫調(diào)節(jié)活性,參與體內(nèi)各種免疫調(diào)節(jié)。近期研究表明,MSCs會分泌大量的細(xì)胞因子,招募CD11b+Ly6C+Ly6G單核細(xì)胞、F4/80+巨噬細(xì)胞和CD11b+Ly6CdimLy6G+中性粒細(xì)胞到腫瘤細(xì)胞周圍加速腫瘤的生長。另外有研究發(fā)現(xiàn),MSCs能夠使單核-巨噬細(xì)胞轉(zhuǎn)化為具有免疫抑制功能的表型,從而促進(jìn)腫瘤進(jìn)展。MSCs可以促進(jìn)中性粒細(xì)胞的存活,但是MSCs對中性粒細(xì)胞功能的確切作用還有待進(jìn)一步闡明。 目的：本研究旨在觀察MSCs對CD11b+Ly6G+中性粒細(xì)胞的作用,揭示MSCs誘導(dǎo)CD11b+Ly6G+中性粒細(xì)胞的免疫抑制功能,促進(jìn)腫瘤進(jìn)程。 方法：(1)將正常小鼠骨髓中或荷瘤小鼠脾臟中分選的CD11b+Ly6G+中性粒細(xì)胞與MSCs共培養(yǎng)72h后,應(yīng)用流式細(xì)胞技術(shù)測定活細(xì)胞百分比與細(xì)胞免疫表型。(2)通過T細(xì)胞抑制性實驗檢測與MSCs共培養(yǎng)后正常小鼠骨髓中或荷瘤小鼠脾臟中CD11b+Ly6G+中性粒細(xì)胞：將與TNF-α刺激過的MSCs或MSCs共培養(yǎng)后CD11b+Ly6G+中性粒細(xì)胞加入抗-CD3抗體刺激的T淋巴細(xì)胞體系中,共培養(yǎng)72h。流式細(xì)胞檢測T淋巴細(xì)胞的增殖和活化。(3)建立4T1腫瘤模型,體內(nèi)檢測與TNF-(I刺激過的MSCs或MSCs共培養(yǎng)后正常小鼠骨髓中或荷瘤小鼠脾臟中CD11b+Ly6G+中性粒細(xì)胞對腫瘤的影響作用。(4)測量與TNF-α刺激過的MSCs或MSCs共培養(yǎng)后小鼠骨髓CD11b+Ly6G+中性粒細(xì)胞中精氨酸酶含量的變化。(5)通過基因芯片分析與TNF-α刺激過的MSCs共培養(yǎng)后正常小鼠骨髓中或荷瘤小鼠脾臟中CD11b+Ly6G+中性粒細(xì)胞中各分子mRNA水平的變化,并通過RT-PCR驗證。(6)通過阻斷與TNF-α刺激過的MSCs共培養(yǎng)后正常小鼠骨髓中或荷瘤小鼠脾臟中CD11b+Ly6G+中性粒細(xì)胞中精氨酸酶和NO,明確MSCs誘導(dǎo)的CD11b+y6G+中性粒細(xì)胞對T細(xì)胞抑制的機制。 結(jié)果：(1)在體外培養(yǎng)過程中,MSCs可促進(jìn)正常小鼠骨髓中或荷瘤小鼠脾臟中CD11b+Ly6G+中性粒細(xì)胞的存活。(2)MSCs誘導(dǎo)正常小鼠骨髓中或荷瘤小鼠脾臟中CD11b+Ly6G+中性粒細(xì)胞對T細(xì)胞的抑制功能。(3)MSCs誘導(dǎo)的正常小鼠骨髓中或荷瘤小鼠脾臟中CD11b+Ly6G+中性粒細(xì)胞在體內(nèi)促進(jìn)腫瘤的生長。(4)MSCs誘導(dǎo)正常小鼠骨髓中CD11b+Ly6G+中性粒細(xì)胞精氨酸酶的升高。(5)通過基因芯片的分析,發(fā)現(xiàn)MSCs可誘導(dǎo)正常小鼠骨髓中或荷瘤小鼠脾臟中CD11b+Ly6G+中性粒細(xì)胞中大量促進(jìn)腫瘤生長的分子上調(diào)。(6)iNOS是MSCs誘導(dǎo)的正常小鼠骨髓中或荷瘤小鼠脾臟中CD11b+Ly6G+中性粒細(xì)胞抑制T細(xì)胞增殖和活化的重要因子。 結(jié)論：MSCs可以通過誘導(dǎo)正常小鼠骨髓中或荷瘤小鼠脾臟中CD11b+Ly6G+中性粒細(xì)胞的免疫抑制作用促進(jìn)腫瘤進(jìn)展
[Abstract]:Background: mesenchymal stem cells (MSCs) is a multipotent stem cell derived from mesoderm with high self-renewal ability and multidirectional differentiation. It exists widely in various tissues of the whole body. It can be cultured in vitro and can be differentiated into nerve cells, osteoblasts, chondrocytes, cardiomyocytes and lipids under specific conditions. Adipose cells, which are the preferred seed cells in cell replacement therapy and tissue engineering, have broad prospects for clinical application. In addition, the karyotype and telomerase activity of mesenchymal stem cells are stable, no expression of histocompatibility complex (MHC) and other stimulators are expressed. Therefore, the host T cells are not easily identified and can escape the rejection of the host immune system. Low immune.MSCs can also inhibit the proliferation of B lymphocyte and T lymphocyte by paracrine effect, induce apoptosis of T lymphocyte and induce immunosuppressive effect. A variety of factors in microenvironment can affect the immune regulation of MSCs, TNF-a can induce MSCs high expression iNOS, and directly inhibit the immune response by the way of NO dependence. It also promotes the up-regulated expression of COX-2 and PGE2 and directly inhibits the immune response through PGE2 dependence. In addition, MSCs can affect the growth of the tumor by producing more monocytes, macrophages and neutrophils by secreting a large number of cytokines. But how does MSCs affect the CD11b+Grl+ cells in the bone marrow, and how this effect is done The use of the tumor process remains to be further explored.
Objective: To investigate the effect of mesenchymal stem cells (MSCs) on the immune function of bone marrow derived CD11b+Grl+ cells.
Methods: (1) the CD11b+Gr1+ cells in mouse bone marrow were co cultured with MSCs for 72h, and the percentage of living cells and cell immunophenotype were measured by flow cytometry. (2) CD11b+Grl+ cells were co cultured with MSCs by T cell inhibition test, and CD11b+Grl+ cells were co cultured with MSCs or MSCs stimulated by TNF- alpha, and CD11b+Grl+ cells were added. In the T lymphocyte system stimulated by anti -CD3 antibody, the proliferation and activation of T lymphocytes were detected by co culture 72h. flow cytometry. (3) the 4T1 tumor model was established, and the effect of CD11b+Grl+ cells on the tumor after TNF- alpha stimulated MSCs or MSCs co culture was detected in vivo.
Results: MSC can promote the survival and formation of CD11b+Grl+ cells in the bone marrow and induce the inhibition of CD11b+Grl+ cells in the bone marrow of normal mice. The results of animal experiments show that the bone marrow CD11b+Grl+ cells treated by MSCs can promote the growth of the tumor and accelerate the death of the mice.
Conclusion: MSCs can accelerate the growth process of tumor by promoting the inhibition of CD11b+Grl+ cells from bone marrow.
Background: neutrophils exist in a large number of tumor microenvironments, but the effect of neutrophils on the progression of the tumor is unknown. Under the action of specific neutrophils, a certain proinflammatory function can be played. Furthermore, neutrophils can be transformed from phenotypes that never have immunosuppressive function to a phenotype with immunosuppressive function: a study has been found. Granulocyte macrophage colony stimulating factor (GM-CSF) and TGF- beta can convert neutrophils from bone marrow from mice to immunosuppressive phenotypes. However, in the process of cancer development, we know little about how neutrophils are converted to immunosuppressive phenotypes and thus promote tumor development.
Mesenchymal stem cell (MSCs) is a kind of regenerative matrix cells in bone marrow, and supports hematopoiesis as the support cells of bone marrow hematopoietic stem cells. Meanwhile, MSCs also has immunoregulation activity and participates in various immunoregulation in the body. In the near future, MSCs secretes a large number of cytokines and recruits CD11b+Ly6C+Ly. 6G mononuclear cells, F4/80+ macrophages and CD11b+Ly6CdimLy6G+ neutrophils accelerate the tumor growth around the tumor cells. Other studies have found that MSCs can convert mononuclear macrophages into immunosuppressive phenotypes, thus promoting tumor progression by.MSCs to promote neutrophils survival, but MSCs against neutrophils The exact role of cell function remains to be elucidated.
Objective: the purpose of this study was to observe the effect of MSCs on CD11b+Ly6G+ neutrophils, and to reveal the immunosuppressive function of MSCs induced neutrophils and promote the progression of tumor.
Methods: (1) after co culture of CD11b+Ly6G+ neutrophils in the spleen of normal mouse bone marrow or tumor bearing mice and MSCs co culture 72h, the percentage of living cells and cell immunophenotype were measured by flow cytometry. (2) CD11b+Ly6G in the spleen of normal mice or the spleen of tumor bearing mice was detected by T cell inhibition test and MSCs co culture. + neutrophils: CD11b+Ly6G+ neutrophils were co cultured with MSCs or MSCs stimulated by TNF- alpha, and CD11b+Ly6G+ neutrophils were added to the T lymphocyte system with anti -CD3 antibody stimulation. The proliferation and activation of T lymphocytes were detected by co culture 72h. flow cytometry. (3) a 4T1 tumor model was established, and TNF- (I stimulated MSCs or co cultured normal mice) The effect of CD11b+Ly6G+ neutrophils in the spleen of bone marrow or tumor bearing mice. (4) the changes in arginase content in bone marrow CD11b+Ly6G+ neutrophils of mice were measured with TNF- alpha stimulated MSCs or MSCs. (5) the bone marrow or charge of normal mice was cultured by gene chip analysis and TNF- alpha stimulated MSCs Co culture. The changes in the mRNA level of each molecule in the CD11b+Ly6G+ neutrophils in the spleen of the tumor mice and verified by RT-PCR. (6) the CD11b+Ly6G+ neutrophil and NO in the spleen of normal mice or the spleen of the tumor bearing mice were inhibited by MSCs co culture with TNF- alpha stimulated MSCs. The CD11b+y6G+ neutrophils induced by MSCs were clearly suppressed to T cells. The mechanism of the system.
Results: (1) in vitro culture, MSCs can promote the survival of CD11b+Ly6G+ neutrophils in the spleen of normal mice or tumor bearing mice. (2) MSCs induced inhibition of T cells by CD11b+Ly6G+ neutrophils in the spleen of normal mice or in the spleen of tumor bearing mice. (3) the spleen of normal mice or the spleen of tumor bearing mice induced by MSCs CD11b+Ly6G+ neutrophils promote tumor growth in the body. (4) MSCs induces the increase of CD11b+Ly6G+ neutrophil arginase in the bone marrow of normal mice. (5) through the analysis of gene chip, it is found that MSCs can induce a large number of molecules that promote tumor growth in the spleen of normal mice or in the spleen of tumor bearing mice, CD11b+ Ly6G+ neutrophils. (6) iNOS is an important factor for MSCs induced CD11b+Ly6G+ neutrophils to inhibit proliferation and activation of T cells in normal mouse bone marrow or tumor bearing mice.
Conclusion: MSCs can promote tumor progression by inducing the immunosuppressive effect of CD11b+Ly6G+ neutrophils in normal mouse bone marrow or spleen bearing mice.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R329.2

【參考文獻(xiàn)】

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1 高麗霞;郭惠芳;嚴(yán)靜霞;孫熠峰;馬毓梅;王志會;靳洪濤;;轉(zhuǎn)錄因子Foxp3在原發(fā)性干燥綜合征中的異常表達(dá)[J];免疫學(xué)雜志;2013年05期

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